Water insoluble helychrisum extract, process for preparing the same and uses thereof

ABSTRACT

The present invention relates to a water insoluble helychrisum extract, a process for preparing the same and its use for preparing pharmaceutical compositions for oral and/or topical administration. Furthermore, the present invention relates to the pharmaceutical compositions including said extract.

The object of the present invention is a water insoluble helychrisumextract, a process for producing the same and the use thereof forpreparing pharmaceutical compositions for oral and/or topicaladministration. Furthermore; the present invention relates to thepharmaceutical compositions including said extract. Helychrisum(Helichrysum italicum G. Don) is a perennial plant belonging to thegenus of the Asters, better known as Composites. It is an aromatic plantoriginating from the Mediterranean region, where it grows on dry sandysoils; its flowers are combined in corymbs and have a typical goldenyellow colour, from which the name of the plant comes from (from theGreek Helios chrysos, golden sun).

The “dry drug” of the helychrisum (hereinafter, “the drug”) consists ofthe dried flowered tops of the plant. By the term “flowered tops of theplant” is understood to mean the flowers plus the tender, non woodytops, of the branchlets, cut at about 5-15 cm from the top, preferablyat about 10 cm, depending on the plant.

Amongst the chemical compounds found in the drug, there have to bementioned the flavonoids, such as, for example: pinocembrin, apigenin,isoquercitrin, naringenin, luteolin, 7-glucoside, gnaphaline;tiliroside; 4,2′,4′,6′-tetrahyroxychalcone-2′-O-glucoside;naringin-4′-O-glucoside; kampferol-3-O-glucoside. Other types ofchemical compounds found in the tops and the flowers of helychrisum are:phthalides (5-methoxy-7-hydroxy-phthalyde and 5,7-dimethoxyphthalide);triterpenes (ursolic acid, uvaol, α-amirine); acetophenone derivatives;caffeil-quinic derivatives; β-sitosterol; nerols and neril acetate(these latter comprise the 30-500 of the essential oils existing withinthe plant in a varying percentage, between 0.05% and 0.20% by weight,based on the weight of the drug, depending on the species and theseason).

The essential oils are generally obtained by steam distillation of theflowered tops of the plant.

The known patent art mainly describes the use of helychrisum essentialoils.

In the US 2005/0003028 patent, the use both of a decoction of a drugmixture, containing the 2% of helychrisum flowers, and an alcoholicextract of a 3:1 mixture of Marian thistle and helychrisum flowers; inboth cases, the addition of helychrisum to the preparation is motivatedfor the detoxifying properties and assistance for the bile secretion. Inthe US 2004/0258783 patent application, the anti-wrinkle action exertedby the essential oil extracted by steam distillation from the floweredtops is claimed.

In the U.S. Pat. No. 5,785,972 patent, a composition containingcommercial helychrisum oil (together with honey, colloidal silver, watersoluble lecithin), to be used in the treatment of burns and sores isclaimed.

In the WO 02/07744 patent application, an association of at least twoessential oils (one of which of helychrisum), to be used for improvingthe resistance to chemotherapeutics in patients under therapy for viralhepatites or tumours, is claimed.

In the WO 03/015522 patent application, the use of an acaricidalcomposition consisting of a mixture of essential oils (among which thehelychrisum) is claimed. In the FR 2830198 patent, the use of acomposition containing helychrisum oil, together with other essentialoils for the topical treatment of viral infections, immunodeficienciesand other pathologies, such as the cystic fibrosis, is claimed.

In the FR 2845594 patent, the use of a composition containinghelychriSum oil, together with other essential oils for the use incosmetics or dermatology, is claimed.

In the FR 2774585 patent, the use of a composition containinghelychrisum oil, together with other essential oils for stopping thehair loss and fighting for the formation of cuticles (dandruff), isclaimed.

In the KR 2003/046949 patent application, the extraction of the oilyfraction of helychrisum angustifolium and the anti-inflammatory activityof a cosmetic preparation are claimed.

The preparations obtained by the helychrisum drug more generally used atpresent, are; the decoction, the fluid extract, the syrup, the total dryextract.

By the term “decoction”, is understood to mean a liquid which is usuallyprepared extemporarily at the time of use by placing an opportune drugquantity in cold water, boiling the water and filtering away the coarseresidue remained in suspension before consuming the extraction liquid.

By the term “fluid extract” (hereinafter “fluid helychrisum extract”) isunderstood to mean the liquid, more or less thick, which is obtained bymacerating the drug with a proper quantity of solvent mixture, normallyhydro alcoholic (alcohol at 60-70° or alcohol at 95°) or hydro glyceric,for a time period varying depending on the kind of drug and,subsequently, filtering the macerated matter in order to eliminate theinsoluble residue of the drug.

By the term “syrup” is understood to mean a liquid which is obtained byproperly diluting the above fluid extract (normally with water) andadding flavours, sugar and excipients of a different nature.

By the term “total dry extract” (hereinafter, “dry helychrisum extract”)is understood to mean the solid residual which is obtained by completelyremoving, by evaporation, the solvent mixture (hydro alcoholic or hydroglyceric) from the above fluid extract, operating at low temperatures.The total dry helychrisum extract can be also prepared in a freeze-driedform (hereinafter, freeze-dried helychrisum extract”, or “extractedfreeze-dried helychrisum” in the enclosed Tables).

Unfortunately, also the above helychrisum preparations, even if they areknown for their good tolerability, are affected by a series of nonnegligible drawbacks, typical of the phyto-therapeutic preparations justdescribed.

For example, the mixture of the extracted active substances (responsiblefor the pharmacological activity/s ascribed to the drug) can result toomuch diluted (as in the case of the fluid extract and/or the syrup) or,even not reproducible, nor standardizable as for the dosage (as in caseof decoction).

Furthermore, the extraction methods above described are not able toselectively extract only the pharmacologically beneficial activesubstances, but, at the same time, they extract a significant quantityof substances at least pharmacologically inert, if not potentially orreally toxic.

Therefore, for the purpose of the optimization of the pharmacologicalactivity of the helychrisum and the standardization of the doSages, inparticular for the treatment of the acute and chronic pathologies, itshould be useful to be able to provide pharmaceutical compositionsincluding a therapeutically effective quantity of helychrisum,characterized by a high quantity of pharmacologically beneficial activesubstances (as a standardized title) and a low quantity ofpharmacologically inactive, if not even toxic, substances. Likewise, itshould be useful to be able to provide pharmaceutical compositionswherein the quantity of mixture including the beneficial activesubstances of the helychrisum (dose) is, beside being standardized inthe components thereof, substantially lower to the one normallyadministered with the traditional preparations above described.

Compositions having the features above described are not known.

Therefore, there remains the need of being able to providepharmaceutical compositions based on helychrisum extract, including atherapeutically effective quantity of the same, containing the greatestpossible quantity of pharmacologically beneficial active substances andthe lowest possible quantity of pharmacologically inactive, if nottoxic, substances.

An object of the present invention is to give an adequate answer to theneed above described.

These and other objects, which will result evident from the followingdetailed description, have been attained by the Applicant, which hasunexpectedly found that a water insoluble fraction of the above fluidhelychrisum extract is able to solve the problem above described.

By easiness, hereinafter said water insoluble fraction will be shown bythe term “water insoluble helychrisum extract” (or “water insolublehelychrisum fraction” in the enclosed Tables).

It is an object of the present invention a water insoluble helychrisumextract, as reported in the appended independent claim.

Another object of the present invention is a process for preparing saidwater insoluble helychrisum extract, as reported in the appendedindependent claim.

Another object of the present invention is then a pharmaceuticalcomposition including the above water insoluble helychrisum extract, asreported in the appended independent claim.

Another object of the present invention is the use of said waterinsoluble helychrisum extract for the preparation of pharmaceuticalcompositions, as reported in the appended independent claim.

Further objects of the present invention are the use of said extract asa medicament and/or for the preparation of pharmaceutical compositionsfor the treatment of pathological states in humans and animals, asreported in the appended claims.

Preferred embodiments of the present invention are reported in theappended dependent claims.

The present invention is shown in detail in the following description.Said invention is further illustrated also with the help of the enclosedTables 1 to 3, wherein:

-   -   Table 1 shows, from the top downwards, the comparison between        the chromatographic profiles of, respectively: a) freeze-dried        helychrisum extract; b) water soluble helychrisum fraction        (namely the fraction of the freeze-dried helychrisum extract        soluble in water); c) water insoluble helychrisum fraction        (namely the water insoluble helychrisum extract according to the        present invention), wherein the chromatograms have been obtained        with the HPLC-MSD method described in the following experimental        section (Example 4)    -   Table 2 shows, from the top downwards, the comparison between        the chromatographic profiles of, respectively: a) freeze-dried        helychrisum extract; b) water soluble helychrisum fraction        (namely the fraction of the freeze-dried helychrisum extract        soluble in water); c) water insoluble helychrisum fraction        (namely the water insoluble helychrisum extract according to the        present invention), wherein the chromatograms have been obtained        with a detector adjusted to 220 nm, with the HPLC-DAD method        described in the following experimental section (Example 4)    -   Table 3 shows, from the top downwards, the comparison between        the chromatographic profiles of, respectively: a) freeze-dried        helychrisum extract; b) water soluble helychrisum fraction        (namely the fraction of the freeze-dried helychrisum extract        soluble in water); c) water insoluble helychrisum fraction        (namely the water insoluble helychrisum extract according to the        present invention), wherein the chromatograms have been obtained        with a detector adjusted to 370 nm, with the HPLC-DAD method        described in the following experimental section (Example 4).

As it has been previously mentioned, the Applicant has unexpectedlyfound that the therapeutic properties of the Helichrysum italicum aresubstantially referable/ascribable to a water insoluble fraction of thefluid helychrisum extract (that is, that fraction previously defined as“water insoluble helychrisum extract”, obtainable from the drug (or fromits fluid extract, or from its total dried/freeze-dried extract) bymeans of a particular and original extraction process.

According to a preferred embodiment of the present invention, the waterinsoluble helychrisum extract is obtainable by means of an extractionprocess from the drug including the following steps:

1) extracting the drug with a water/organic solvent mixture in order togive the hydro-organic fluid tract of helychrisum;2) evaporating the organic solvent from said fluid extract, to give theprecipitation of a water insoluble fraction from the remaining aqueousfraction;3) separating said water insoluble fraction from said aqueous fraction,to give a water insoluble solid extract.

When one wishes to start from the total dried/freeze-dried extract,instead of the drug or the fluid extract, then the steps 1) and 2)should be replaced by the preparation of a suspension of said totaldried/freeze-dried extract in a proper quantity of water.

In the step 1), the water/organic solvent mixture has a water content,relative to the total volume of the mixture, from 15% (v/v) to 85%(v/v); preferably, from 20% (v/v) to 65% (v/v); more preferably,approximately equal to 30% (v/v).

The organic solvent employed in said mixture is a partly or completelywater soluble solvent, preferably selected from acetone and an alcoholcontaining from one to three carbon atoms (methanol, ethanol,n-propanol, isopropanol), or their mixtures; more preferably, saidorganic solvent is ethanol.

In a preferred embodiment of the invention, said water/organic solventmixture consists of 70° ethanol.

In this case, in particular, the ethanol is produced from biologicalwheat and consists of 66.12 parts (by weight) of alcohol against 33.88parts (by weight) of water.

In the step 2), the evaporation of said organic solvent is preferablycarried out under a reduced pressure; preferably, at a residual pressurefrom 600 mbar to 150 mbar.

Preferably, the evaporation is then continued until the initial volumeof the fluid extract coming from the passage 1) has been reduced at avalue from about ⅓ to about 1/7 of said initial volume; preferably, fromabout ¼ to about ⅙ of the initial volume; more preferably, at about ⅕ ofthe initial volume.

In the step 3), the separation of said water insoluble fraction from theaqueous fraction is preferably carried out by centrifugation orfiltration; more preferably, by centrifugation.

In a preferred embodiment of the invention, said extraction processfurther includes the step of:

4) drying the water insoluble extract coming from the above passage 3).

In the step 4), the drying is carried out in such conditions to avoidthe degradation of said extract; preferably, said drying is carried outby freeze-drying; preferably, of an aqueous suspension of the extractitself.

The above step 1) (that is the preparation of the hydro-organic fluidextract of helychrisum) has the object of extracting the greaterpossible number of pharmacologically active substances existing in thedrug.

In a preferred embodiment of the invention, said step includes at leastone of the following steps (preferably all of them):

1a) extracting (by maceration and percolation) the drug, using adrug/solvent mixture (D/S) weight ratio (w/w) from 1:1 to 1:25;preferably, from 1:5 to 1:20; more preferably, from 1:10 to 1:15; stillmore preferably, of about 1:13;1b) extracting the drug a a temperature from 20° C. to the refluxtemperature of the organic solvent used, or the water/organic solventmixture; preferably, at a temperature of 60° C.;1c) extracting the drug in two steps: namely, by carrying out a firstextraction, preferably with about 60% of the solvent mixture, followedby a second extraction, with the remainder about 40%, and joiningtogether the two extracts;1d) filtering the end fluid extract for the purpose of clarifying thesame.

The above step 2) (that is the partial evaporation of the hydro-organicsolvent mixture) has the purpose of completely removing the organicsolvent from the fluid extract deriving from the step 1) and partlyconcentrating the water until the water insoluble fraction (namely, thewater insoluble helychrisum extract), which contains the mixture of thepharmacologically beneficial active substances of the drug, hasprecipitated.

In a preferred embodiment of the invention, said step 2) includes atleast one of the following steps (preferably all of them):

2a) evaporating the organic solvent under a reduced pressure; preferablyat a residual pressure from 600 mbar to 150 mbar;2b) carrying out said evaporation at a outer temperature from 40° C. to130° C.; preferably, from 50° C. to 70° C.; more preferably, at about60° C.

The above step 3) (namely the separation of the water insoluble extractof helychrisum from the aqueous step) has the putpose of isolating andwashing the helychrisum extract from the residual aqueous fraction,which includes a series of non pharmacologically active water solubleproducts.

In a preferred embodiment of the invention, said step 3) includes atleast one of the following steps (preferably all of them):

3a) centrifuging the solid/water phase mixture resulting from thepassage 2), for example by means of a horizontal centrifuge;3b) collecting the solid (that is the water insoluble extract ofhelychrisum);3c) washing said solid with water; for example, using a solid/waterweight ratio (w/w) from 1:1 to 1:10; preferably, from 1:1 to 1:5.

The above additional, possible, passage 4) has the purpose oftransforming the isolated and washed water insoluble helychrisum extractin a powder utilizable for preparing and standardizing the desireddifferent pharmaceutical formulations based on helychrisum.

In a preferred embodiment of the invention, said step 4) includes:

4a) suspending the solid helychrisum extract in water;4b) freezing said suspension;4c) freeze-drying said frozen suspension;4d) milling the obtained freeze-dried product for the purpose ofrendering homogeneous the particle size of solid freeze-driedhelychrisum extract.

The above freeze-drying process is carried out using equipments andworking conditions known to a skilled in the art.

The solid helychrisum extract can also be freeze-dried in the presenceof proper excipients, such as, for example: stabilizers, preservatives,flavourings, sweeteners.

In an embodiment of the invention, the Italic helychrisum used for thepreparation of the pharmacologically active water insoluble extract, hasbeen cultivated in a calcareous and half-arid land typical of theTuscany-Emilia Apennine. The cultivation, arranged at 600 metres abovethe sea-level, was exposed to South-South-West, with an optimumexposition to the sun's rays and a slope about 15-200. The harvestinghas been carried out in July, at the beginning of the blossom time,cutting the tender tops of the branches for 10 cm about and avoiding thelower wooden portion. The fresh harvest has been immediately dried inair furnaces at a temperature of 40-45° C. The dried drug thus obtainedhas shown a flavonoid content, expressed as isoquercitrin, from 0.42% to0.50% by weight, based on the total weight of the drug.

Furthermore, a content in caffeil-quinic derivatives, expressed aschlorogenic acid (monoester of the quinic acid with the caffeic acid),from 2.5% to 3.1% by weight, based on the total weight of the drug, hasbeen determined.

As pointed out in the enclosed Table 1 (TAB. 1—chromatogram a)), thechromatographic profile of the total freeze-dried helychrisum extract,obtained from the drug by the HPLC-MSD method, described hereinafter inthe Example 4, is characterized by the presence of a series ofsignals/products having widely varying retention times (from few minutesto about 40 minutes, until a series of signals having retention timeslower than about 60 minutes).

The chromatographic profile of the water soluble fraction of thehelychrisum extract (TAB. 1—chromatogram b)) is, on the contrary,characterized by the presence of the series of signals having retentiontimes lower than about 40 minutes.

In turn, the chromatographic profile of the water insoluble extract ofhelychrisum (TAB. 1—chromatogram C)) is, on the contrary, characterizedby the presence of the series of signals having retention times higherthan about 60 minutes.

The same type of situation is also substantially confirmed by thechromatograms of the enclosed Tables 2 and 3, from which it is evidentthat the (active) substances, forming the water insoluble helychrisumextract are characterized by retention times higher than about 40minutes (differently from the total freeze-dried total and the watersoluble fraction).

The water insoluble helychrisum extract corresponds to about 2.3%-2.5%by weight with respect to the total weight of the dry drug and about13%-15% by weight with respect to the total weight of the total dryextract of the same.

The water insoluble helychrisum extract is characterized by a watersolubility (at a room temperature of 25° C.)<10 mg of extract/10 ml ofwater; preferably, lower than 8 mg/10 ml; more preferably, lower than 5mg/10 ml.

The solubility study of said extract in other solvents has allowed toindividuate the following solubility profile (expressed as mg of waterinsoluble extract/ml of solvent):

ethanol 95° about 10 mg/0.2 ml;ethyl acetate about 10 mg/0.5 ml;ethyl ether<10 mg/lchloroform about 10 mg/0.5 ml;dichloromethane about 10 mg/l ml;hexane<10 mg/2.5 ml;toluene about 10 mg/l ml;acetone about 10 mg/0.8 ml.

The water insoluble helychrisum extract, obtained according to theextraction method of the present invention, has been additionallycharacterized by chemical and biological methods, for the purpose ofstandardizing the same not only from the constituents point of view butalso the pharmacological activity:

As for the qualitative characterization of the extract, chromatographicmethods have been developed by means of HPLC/DAD and HPLC/MSD, asdescribed hereinafter in the Example 4, which have allowed to acquirethe characteristic chromatographic profile (fingerprint) of the extractitself.

As already above reported, the water insoluble helychrisum extractresulted characterized by the chromatographic profiles (fingerprint) c)disclosed in enclosed TAB. 1-3.

As for the quantitative characterization of said extract; the same hasbeen carried out through the determination of two important classes ofconstituents, the flavonoids and the caffeic-quinic derivatives.

The spectrophotometric methods employed for carrying out suchquantitative determinations are described in detail in the followingexperimental section (Examples 2 and 3, respectively).

The water insoluble helychrisum extract resulted characterized by aflavonoid content, expressed as isoquercitrin, from 2% to 15% by weight,with respect to the total weight of the extract; preferably, from 2.5%to 10% by weight; more preferably, from about 3% to about 8% by weight.

On average, said flavonoid content is from about 3.5% to 5.5% by weight;preferably, around 4% eight. Therefore, in the water insoluble extract,the flavonoids are resulted more concentrated with respect to theirstarting concentration in the dry drug.

Furthermore, the water insoluble helychrisum extract is resultedcharacterized by a content of caffeil-quinic derivatives, expressed aschlorogenic acid, from 0.5% to 5% by weight, with respect to the totalweight of the extract; preferably, from 2.86% to 3.5% by weight; morepreferably, from 2.9% to 3.4% by weight; on average, of about 3% byweight.

The water insoluble helychrisum extract has also been characterized bydetermining its protection activity from the proteinic denaturation,expressed as EC₅₀.

Said extract has proven to be standardized to inhibit a 50% proteinicdenaturation when used at a concentration of 0.2 mg/ml.

The pharmacological activities ascribed to the traditionally usedextracts of the helychrisum, have resulted referable/ascribable to themixture of compounds contained in the water insoluble helychrisumextract obtained through the innovative extraction/separation process ofthe present invention.

The anti-inflammatory/pain-killing activity of said extract has beendocumented, at a pre-clinical level, using the in vitro test of theproteinic denaturation and of the Writhing in the mouse.

The anti-allergic/anti-asthmatic activity has further been documented onthe bronchospasm and the plasma extravasation induced by ovalbumin inguinea pigs sensitized to this compound.

The studies have confirmed that the pharmacological activity of thewater insoluble helychrisum extract, obtained by theextraction/separation process of the present invention, is the one knownof the whole drug. Moreover, the pharmacological activity of saidextract has resulted greater and better than the one ascribable to thedrug, thus allowing to prepare pharmaceutical compositions with areduced and standardized dosage which allow the repeatability of thedosages and the treatments.

Advantageously, the water insoluble helychrisum extract has shown topossess a good anti-inflammatory activity.

Furthermore, said extract has a good pain-killing activity.

Furthermore, said extract has a good anti-allergic activity.

Therefore, the water insoluble extract of the present invention hasproved to be particularly useful for a use as a medicament; inparticular for preparing pharmaceutical compositions to be used in amedical field in the therapy of pathologic states in human and animal,as above pointed out.

Therefore, it is also a subject of the present invention a process forthe preparation of a pharmaceutical composition containing atherapeutically effective quantity of water insoluble helychrisumextract; said process includes at least a step in which said extract isadditioned with opportune pharmacologically acceptable co-formulations.

Said pharmaceutical compositions can be used both for oral and topicaladministration (including aerosol).

Said pharmaceutical compositions are preferably formulated in admixturewith appropriate excipients, such as vehicles, lubricants, dispersants,flavourings, sweeteners, stabilizers, preservatives, antioxidants,additives, such as amino acids, vitamins, enzymes commonly used in thepharmaceutical formulation art.

By mere way of absolutely not limiting example, amongst the particularlypreferred excipients and additives there may be mentioned starch,flavours, such as those of mandarin, grapefruit, strawberry, bilberry,all fruits, sucrose, glucose, ascorbic acid, glutamine, arginine,inulin.

Particularly preferred compositions of the present invention are thosefor oral administration (including the sublingual one).

Typical preferred formulation forms are, for example, capsules, beads,syrups, solutions or suspensions ready-to drink, powders or granulatesin sachets (to be suspended or dissolved in water or in non-carbonatedand non-alcoholic beverages at the moment of use) or analogous forms,tablets, effervescent formulations:

The compositions of the present invention can also be formulated in acoated, lacquered, encapsulated or microencapsulated form, such that toresult gastro-resistant.

Said compositions can also be formulated in a controlled-release form,so as to selectively deliver the active substances in the intestinaltract, in particular within the colon.

Other preferred compositions of the invention are those for topicaladministration.

Said compositions can be prepared in form of pomades, ointments, gels;or they can be opportunely formulated and incorporated in transdermalvehicles, such as for example plasters.

The compositions of the present invention are prepared in a traditionalway by using, depending on the kind of formulation that one wishes tocarry out, preparative techniques known to the pharmaceutical artisanskilled in the art.

The compositions of the present invention have proved to be particularlyuseful for the prevention and/or the treatment of different pathologies,both for topical use (for example for the treatment of eczema and atopicdermatitis, irritative contact dermatitis, allergic contact dermatitis),and for systemic use (for example chronic rheumatic affections,inflammatory pathologies, acute pain from fractures consequences, asthmaand allergic rhinitis).

The present invention is described hereinafter, by mere way of nonlimiting example, by means of some experimental examples.

EXAMPLE 1

Preparation of the water insoluble extract of italic helychrisum,starting from the drug.

178 kg of dry drug of helychrisum were extracted with 70° ethanol withthe D/S weight ratio 1:13. A first extraction with the 600 of thesolvent mixture was carried out over 5 h at 60° C., then the hydroalcoholic extract was removed and the drug extracted a second time withthe remaining 40% of the solvent mixture for 3 h at 60° C. Theextraction was carried out by percolation/maceration.

The pooled hydro alcoholic extracts were filtered (weight of the fluidextract=1830 kg) and concentrated under vacuum (60° C., 600 mbar) until380 kg of a concentrated aqueous suspension were obtained.

The concentrated aqueous suspension was subjected to centrifugationthrough a horizontal centrifuge. In this way, the precipitate wasseparated from the remaining water phase in form of a black-colouredthick, solid mass.

6 kg of wet solid precipitate and 370 kg of water phase were thusobtained.

The wet precipitate obtained was washed with 6 l of ultra-neat water,then subjected to freeze-drying giving 4.2 kg of water insolublehelychrisum extract, equal to about 2.4% by weight, based on the initialweight of the drug employed.

The water phase was, in turn, subjected to freeze-drying giving 26 kg ofa water soluble residue, or fraction, equal to about 14.6% by weight,based on the initial weight of the drug employed.

The freeze-dried water insoluble extract was milled by a cryo-mill togive 3 kg of a powder which was stored at a temperature of +4° C., in asealed and under vacuum aluminium sachet. The freeze-dried waterinsoluble extract resulted characterized by a content of flavonoids,expressed as isoquercitrin, equal to 3.78% by weight, based on the totalweight of the extract, and a content of caffeic-quinic derivatives,expressed as chlorogenic acid, equal to 3.36% by weight, based on thetotal weight of the extract.

The chromatographic profiles of the water insoluble extract thusobtained are those respectively reported in the chromatograms c) of theenclosed TAB. 1-3.

EXAMPLE 2

Spectrophotometric method for the determination of the flavonoid content(expressed as isoquercitrin) in the drug and the water insolublehelychrisum extract (modification of the method described in Eur. Ph, vEd., monograph of Sambuci flos)

About 0.60 g of pulverized drug (or about 0.30 g of water insolublehelychrisum extract) were treated with 1 ml of aqueous solution (5 g/l)of hexamethyltetramine, additioned with 20 ml of acetone and 2 ml of ahydrochloric acid solution (70 g of conc. hydrochloric acid in 100 ml ofwater); the suspension was heated until boiling for 30 minutes.

A filtration through cotton in a 100 ml volumetric flask is carried out.The residue and the cotton were treated in two stages each time with 20ml of refluxed acetone over 10 minutes; the two washes were added to theparent solution and, after cooling, adjusted to volume (100 ml) withacetone (Solution A).

20 ml of the solution A (10 ml in case of the water insoluble extract)were added to 20 ml of water and extracted with ethyl acetate (1×15 ml,3×10 ml).

The pooled extract were washed with water (2×50 ml), dried over drysodium sulphate and adjusted to volume with ethyl acetate in a 50 mlvolumetric flask (Solution B).

10 ml of the solution B were transferred in a 25 ml flask and added with1 ml of aluminium trichloride solution (at 20 in methyl alcoholcontaining 5% of glacial acetic acid) and adjusted to volume with themethanol solution (50 ml/l) of glacial acetic acid. (Solution underexamination). 10 ml of the solution B were transferred in a 25 ml flaskand adjusted to volume with the methanol solution (50 ml/l) of glacialacetic acid. (Comparison solution).

After 30 minutes, the absorbance of the solution under examination wasmeasured, at 425 nm, with respect to the comparison solution.

Knowing that A_(1%, 1cm) of the isoquercitrin is equal to 500 at 425 nm,the percent content of flavonoids, expressed as isoquercitrin, wascomputed with the formula:

%=(A×V×F)/A _(1%, 1cm) ×p

wherein:p=weight of the sample expressed in gramsA=absorbance of the sample at 425 nmV=extraction volume (100 ml)F=dilution factor (6.25)

EXAMPLE 3

Spectrophotometric method for the determination of the caffeil-quinicacids content (expressed as chlorogenic acid) in the drug and the waterinsoluble helychrisum extract (isolation of the orthodiphenolic fractionin form of lead salt) (modification of the method described in FU IX ed,monograph of the Cynara dry extract).

About 0.50 g of pulverized drug (or 0.30 g in case of the waterinsoluble helychrisum extract) were treated with 40 ml of water untilboiling. The suspension was heated until boiling, filtered throughcotton in a centrifuge tube. To the still hot solution, 2 ml of asolution saturated with lead (PbII) acetate were added. Cooling andcentrifugation were performed, and the clear supernatant solution waseliminated. The precipitate was washed with water (5 ml), then againcentrifuged removing the water phase. The precipitate was dissolved in70 ml of a 10% acetic acid aqueous solution and heated to ebullition.The hot mixture was filtered through cotton, 2 ml of 20% sulphuric acidwere added. The lead sulphate precipitate was separated bycentrifugation, the clear solution was trans-ferred in a 100 mlvolumetric flask. The precipitate remained on the bottom of thecentrifuge tube was washed with 5 ml of 10% acetic acid; the washingsolution, after centrifugation, was transferred in the same flask, whereit was adjusted to an end volume of 100 ml with 10% acetic acid.

1 ml of this solution was diluted to 25 ml with methanol: the absorbanceof the solution at 325 nm was read, against a solution obtained bydiluting 1 ml of 10% acetic acid to 25 ml with methanol.

Knowing that A_(1%, 1cm) of the chlorogenic acid at 325 nm is equal to485, the percent content of caffeic-quinic acids, expressed aschlorogenic acid, was computed with the formula:

%=(A×V×F)/A _(1%, 1cm) ×p

wherein:A=absorbance of the sample at 425 nm.F=dilution factor (25)p=weight of the sample expressed in gramsV=extraction volume (100 ml)

EXAMPLE 4

HPLC/DAD and HPLC/MSD methods for the acquisition of chromatographicprofile of the helychrisum extracts (applicable, respectively, over:drug, freeze-dried extract, water insoluble extract and water solublefraction)

The study of the HPLC chromatographic profiles was carried out byacquiring the chromatograms by means of two different detectors (DAD:Photo Diode Array; MSD: Ion Trap, Agilent, mod SL) with differentanalytical methodologies in the two cases.

HPLC/DAD Method Equipment:

HPLC Agilent series 1100, fitted with vacuum degasser, quaternary pump,self-sampler, thermostated compartment at 20° C. for the housing of twocolumns, diode array detector (DAD).Reverse phase analytical column Prodigy RP-18 (250 mm×4.6 mm, 100 Å, 5μ,Phenomenex, USA) with a pre-column RP-18 (4 mm×3 mm).

Preparation of the Solutions to be Examined:

-   -   drug: extracting 0.5 g of dry drug with two subsequent portions        of 25 ml of 50° ethanol, over 30′ in an ultrasonic bath;        centrifugation is carried out, the extracts are pooled and        adjusted to volume to 50 ml in a volumetric flask;    -   freeze-dried extract: extracting 0.1 g of extract with 10 ml of        50° ethanol, over 30′ in an ultrasonic bath; centrifugation is        carried out and the overlying solution is examined, as described        in the Process;    -   water insoluble extract (or water insoluble fraction):        extracting 0.1 g with 10 ml of 50° ethanol, over 30′ in an        ultrasonic bath, centrifugation is carried out and the overlying        solution is examined, as described in the Process.    -   water soluble fraction: extracting 0.1 g with 10 ml of 50°        ethanol, over 30′ in an ultrasonic bath, centrifugation is        carried out and the overlying solution is examined, as described        in the Process.

Process:

collecting 0.5 ml of the solution to be examined, filtering over acellulose acetate filter (0.45 μm), injecting 20 μl in the apparatus.Flow rate: 1 ml/minElution solvent: 0.2% H₃PO₄ in water/methyl alcohol, in a gradient(Table no. 1)

TABLE no. 1 Gradient composition: minutes 0.2% H₃PO₄ in water CH₃OH 090% 10% 35 50% 50% 75  5% 95%

The chromatograms are acquired at 220, 265, 280 330 and 370 nm.

HPLC/MSD Method Equipment:

HPLC Agilent series 1100, fitted with vacuum degasser, binary pump,self-sampler thermostated at 10° C., compartment thermostated at 20° C.for the housing of two columns, diode array detector (DAD) and ion trapmass detector, model SL.Reverse phase analytical column Prodigy RP-18 (250 mm×4.6 mm, 100 Å, 5μ,Phenomenex, USA) with a pre-column RP-18 (4 mm×3 mm).

Preparation of the Solutions to be Examined:

-   -   drug: extracting 0.5 g of dry drug with two subsequent portions        of 25 ml of 60% methanol, over 30′ in an ultrasonic bath;        centrifugation is carried out, the extracts are pooled and        adjusted to volume at 50 ml in a volumetric flask, always with        605 methanol;    -   freeze-dried extract: dissolving 10 mg in 10 ml of 60% methanol        over 30′ in an ultrasonic bath;    -   water insoluble extract (or water insoluble fraction):        dissolving 10 mg in 10 ml of methanol, over 30′ in an ultrasonic        bath;    -   water soluble fraction: dissolving 10 mg in 10 ml of 0.60%        methanol over 30′ in an ultrasonic bath.

Process:

collecting 0.5 ml of the solution to be examined, filtering over acellulose acetate filter (0.45 μm), injecting 5 μl in the apparatus.Flow rate: 1 ml/minElution solvent: 0.025% formic acid in water/methyl alcohol, in agradient (Table no. 2)

TABLE no. 2 Gradient composition: 0.025% HCO₂H in minutes water CH₃OH 090% 10% 35 50% 50% 75  5% 95% 80  5% 95%

Before the introduction within the mass detector, the flow is properlysplit to 0.6 ml/min. through a T-valve using, for the differentconnections, capillary tubes with a same caliper but different length.The analyses have been carried out using the ESI source and selectingnegative ions. In Table no. 3, the parameters for the acquisition of themass spectra during the chromatographic elution are reported.

TABLE NO. 3 Acquisition modes of the mass spectra Source ESI Nebulizer50 Capillary 3500 Polarity Negative Dry gas 10 Skimmer −40.0 Target30000 Dry Temp. 350 Cap. exit −90.8 MAT  100 Oct. RF 108.9 Oct 1DC−12.00 Scan 100/1500 Lens 1 5.0 Oct 2DC −1.70 Average 7 (RA = 2) Lens 260.0 Trap drive 55.00

For the water insoluble extract, a specific HPLC/MSD method was furtherdeveloped, capable of pointing out with a greater accuracy thecharacteristic components. The methodology is differentiated from thepreceding one only for the used gradient, whose composition is reportedin the Table no. 4.

TABLE no. 4 Gradient composition 0.025% HCO₂H in minutes water CH₃OH 030%  70% 75 5% 95% 80 5% 95%

Control of the Anti-Denaturant Activity

The test is carried out in vitro, using electrophoresis techniques, andallows to correlate the anti-denaturant activity exerted by the waterinsoluble helychrisum extract on plasma proteins and expressed as EC₅₀,to the anti-inflammatory activity of vegetal extracts. In the same test,in fact, the FANS are capable of inhibiting the denaturation of theproteins.

1-22. (canceled)
 23. Water insoluble helychrisum extract, characterizedby a flavonoid content, expressed as isoquercitrin, from 2% to 15% byweight, based on the total weight of the extract.
 24. Extract accordingto claim 23, wherein said flavonoid content is from 2.5% to 10% byweight, based on the total weight of the extract; preferably, from about3% to about 8% by weight; more preferably, from about 3.8% to about 4.2%by weight.
 25. Extract according to claim 23, further characterized by acontent of caffeil-quinic derivatives, expressed as chlorogenic acid,from 0.5% to 5% by weight, based on the total weight of the extract;preferably, from about 2.8% to 3.5% by weight; more preferably, from2.9% to 3.4% by weight.
 26. Extract according to claim 23, furthercharacterized by a water solubility, expressed by weight ofextract/water volume, <10 mg/ml; preferably, lower than 8 mg/10 ml; morepreferably, lower than 5 mg/10 ml.
 27. Extract according to claim 23,further characterized by a protection activity from the proteinicdenaturation, expressed as EC₅₀, at a concentration of 0.2 mg/ml. 28.Extract according to claim 23, further characterized by the HPLC-MSDchromatographic profile of the chromatogram c) reported in the TAB. 1and/or the HPLC-DAD chromatographic profile, at 220 nm, of thechromatogram c) reported in the TAB. 2 and/or the HPLC-DADchromatographic profile, at 370 nm, of the chromatogram c) reported inthe TAB.
 3. 29. Process for the preparation of a water insolublehelychrisum extract according to claim 23, including the followingsteps: 1) extracting the drug with a water/organic solvent mixture inorder to give the hydro-organic fluid helychrisum extract; 2)evaporating the organic solvent from said hydro-organic fluid extract,to precipitate a water insoluble fraction from the remaining aqueousfraction; 3) separating said water insoluble fraction from said aqueousfraction, to obtain a water insoluble solid extract.
 30. Processaccording to claim 29, wherein, in the step 1), said water/organicsolvent mixture has a water content, based on the total weight of themixture, from 15% (v/v) to 85% (v/v); preferably, from 20% (v/v) to 65%(v/v); more preferably, approximately equal to 30% (v/v); preferably,said mixture consists of 70° ethanol.
 31. Process according to claim 29,wherein, in the passage 1), said organic solvent is a partly orcompletely water soluble solvent selected from acetone and an alcoholcontaining one to three carbon atoms, or their mixtures.
 32. Processaccording to claim 31, wherein said alcohol is selected from methanol,ethanol, n-propanol, isopropanol; preferably, ethanol.
 33. Processaccording to claim 29, wherein, in the passage 2), said evaporation ofthe organic solvent is carried out under a reduced pressure; preferably,at a residual pressure from 600 mbar to 150 mbar.
 34. Process accordingto claim 33, wherein said evaporation is continued until the initialvolume of the fluid extract coming from the passage 1) has been reducedat a value from about ⅓ to about 1/7 of said initial volume; preferably,from about ¼ to about ⅙ of the initial volume; more preferably, at about⅕ of the initial volume.
 35. Process according to claim 29, wherein, inthe passage 3), said separation of the water insoluble fraction fromsaid aqueous fraction is preferably carried out by centrifugation or byfiltration; more preferably, by centrifugation.
 36. Process according toclaim 29, further including the passage of: drying said water insolubleextract coming from the passage 3) of said claim
 29. 37. Processaccording to claim 36, wherein said drying passage is carried out insuch conditions to avoid the extract degradation; preferably, saiddrying is carried out by freeze-drying.
 38. Pharmaceutical compositionincluding an effective quantity of a water insoluble helychrisum extractaccording to claim
 23. 39. Process for the preparation of a compositionaccording to claim 38, including at least a step in which said extractis additioned with pharmaceutically acceptable co-formulations. 40.Method for the treatment of inflammatory pathologies of the human oranimal body comprising the step of administering a pharmaceuticalcomposition comprising an effective dose of the water insolublehelychrisum extract according to claim 23, to a patient in need thereof.41. Method according to claim 40, in which said administration is anoral or topical administration.
 42. Method for the treatment of painfulpathologies of the human or animal body, comprising the step ofadministering a pharmaceutical composition comprising an effective doseof the water insoluble helychrisum extract according to claim 23, to apatient in need thereof.
 43. Method for the treatment of allergicpathologies of the human or animal body, comprising the step ofadministering a pharmaceutical composition comprising an effective doseof the water insoluble helychrisum extract according to claim 23, to apatient in need thereof.